1. Field of the Invention
The present invention relates to liquid delivery devices. More particularly, the present invention relates to air displacement liquid delivery systems that include a liquid delivery device. Even more particularly, the present invention relates to air displacement liquid delivery systems that use air to separate liquid aspirated by the liquid delivery device from one or more components of that liquid delivery device. Still more particularly, the present invention relates to air displacement liquid delivery systems that sense information regarding conditions within and external to a liquid delivery device of the system and that use this information to control liquid displacement by that liquid delivery device. Still more particularly, the present invention relates to methods of using the liquid delivery systems of the present invention.
2. Description of the Prior Art
Present air displacement liquid delivery devices, such as, for example, the device described in PCT Patent Application PCT/US04/03824, which is incorporated herein by reference in its entirety, may be manual or they may be automated. Present manual air displacement liquid delivery devices are configured in the same general way. Referring to FIG. 1, the basic elements of an example manual air displacement liquid delivery device 1 are a piston 2 having a plunger 3 movable within a cylinder 4 which causes air (or other gas), represented by molecule 5, to be displaced along a shaft 6 and into a tip 7, which is able to hold a liquid 8 for delivery of that liquid 8. The point at which the air 5 and liquid 8 meet in the tip 7 is identified herein as interface 5′.
Manual air displacement liquid delivery devices, such as device 1, typically use the air 5 to separate the liquid 8 being delivered from the tip 7 from contacting the shaft 6, the cylinder 4, the plunger 3, the piston 2 and other rigid components of these devices 1. The air 5 therefore serves to prevent contamination and degradation of the delivery device 1 itself, and to prevent contamination of any liquids 8 that are to be aspirated into or delivered from the device 1.
Present automated air displacement liquid delivery devices are configured somewhat differently than are existing manual devices 1. An example of an automated air displacement liquid delivery device is shown in FIG. 2. Automated air displacement liquid delivery device 10 includes the piston 2 having the plunger 3 movable within the cylinder 4. When the piston 2 is moved, air 5 is displaced within flexible tubing 11, within the shaft 6, which is connected to the tubing 11, and within the tip 7. The cylinder 4 and tubing 11 of these automated devices 10 includes a “working fluid” 13 which creates a second air interface 5″ that helps ensure separation of the liquid 8 contained in the tip 7 and the working fluid 13. Further, the piston 2 of the device 10 is actuated with an actuator 14, which in turn is under control of a control unit 15. The device 10 also may include a second actuator 16 used to move the tip 7 to and from vessels for aspiration and dispensation.
When the tip 7 of either type of liquid delivery device is inserted into a source vessel containing a liquid, the liquid seals the opening of the tip 7. When this happens, the mass of the air 5 trapped inside the tip 7 cannot change, except due to evaporation of the liquid. During aspiration, when the liquid 8 enters the tip 7, it will begin to evaporate unless the air 5 above the liquid 8 is already saturated with the vapor. Heat will also be transferred into or away from the air 5 if it is at a different temperature than that of the liquid 8 or the delivery device 1 or delivery device 10. Once the tip 7 is removed from the source vessel, surface tension may hold liquid 8 within the tip 7 even if the gas pressure at the air interface 5′ changes slightly due to continuing evaporation or changing temperature. If surface tension is low due to the type of liquid 8 being aspirated, or if the pressure change in the air interface 5′ is great enough, then liquid 8 may come back out of the tip 7 as it is moved from the source vessel to a receiving vessel, for example.
Further, liquid 8 evaporates from the interface 5′ inside the tip 7 during the aspiration process and any subsequent pause that may occur before the tip 7 is removed from the liquid in the source vessel. According to the ideal gas law, the number of moles of air 5 trapped in the space above the liquid 8 increases due to that evaporation. Thus, the volume of liquid 8 drawn up into the tip 7 is less than would be expected based on the volume of air 5 displaced by movement of the plunger 3. The size of the discrepancy depends on a number of factors; among them are the length of time that the tip 7 remains immersed in liquid contained in the source vessel during aspiration and the vapor pressure of the liquid 8. In the case of liquids with very high vapor pressure (e.g., methanol), the error due to evaporation can be substantial. In these cases, the faster that the tip 7 is withdrawn from the source vessel, the less evaporation can influence the amount of liquid aspirated.
When the delivery device is being calibrated with water as the liquid, it is recommended in International Organization for Standardization (ISO) document ISO 8655, part 6, that it be preconditioned by aspirating and dispensing to waste five aliquots of liquid with the same tip to precondition (humidify) the air (within the shaft or conduit), and it is further recommended that the tip then be replaced, with the liquid then being aspirated and dispensed to waste once more. Further, according to Annex B of ISO 8655, part 2, an error of up to 2% can be incurred if the device is inadequately pre-conditioned (assuming the liquid 8 being delivered is water). Unfortunately, the preconditioning process is time-consuming, and therefore few users of these devices actually follow this procedure for routine liquid deliveries.
The process of preconditioning the device to increase humidity carries with it a liability if the liquid is at a different temperature than the air 5 at the interface 5′. If the aspirated liquid is at a different temperature than the air 5 at the interface 5′, heat will be transferred between the liquid and the air, thereby changing the temperature of the air. (Heat transfer can also take place between the material of one or more components of the device and the air 5 at the interface 5′.) According to the ideal gas law, which states that the volume of a gas (air) is dependent upon its temperature, the volume of liquid aspirated into the tip therefore will be varied when such a change in temperature of the air occurs. This error becomes increasingly greater the longer that the device, including the tip, and the air in the device are exposed to the liquid at the different temperature. Thus, preconditioning of these devises can introduce error due to heat transfer if the liquid has even a slightly different temperature than the device. According to Annex B of ISO 8655 part 2, the possible error due to temperature variations is given as 0.3% per ° C.
For existing liquid delivery devices, liquid entering the tip during aspiration has momentum that keeps it from stopping instantaneously when the plunger stops. Therefore, the liquid keeps flowing for a short time after the plunger stops, thereby compressing the air 5 at the interface 5′. This compression is followed by a rebound of the liquid which causes liquid to be expelled from the tip. After a period of oscillation, equilibrium is established. The magnitude of the oscillation and time it takes it to subside are dependent on the design of the device, including the design of the tip. (Generally, this is a more significant issue for larger volumes, e.g., 1000 μL, and liquids with low viscosity.) The tip should remain immersed in the source vessel during this oscillation period to get the most accurate aspiration of liquid.
The liquid being aspirated or dispensed enters or exits the tip over a period of time, with the duration of that time depending on the viscosity of the liquid. During the time that the liquid is flowing into the tip, the pressure at the interface 5′ is changing toward equilibrium; when equilibrium is achieved, the flow ceases. The flow rate slows as the tip fills due to a decrease in the differential pressure driving the flow. FIG. 3 is a graph of actual aspirated volume of anhydrous glycerol versus time in an application using a previously configured liquid delivery device. The actual aspirated volume 20 of the anhydrous glycerol approaches the final, desired volume 21 (to which the device was calibrated to aspirate) exponentially as endpoint 22 is approached. For a liquid with low viscosity, such as water, the delay before the desired volume is aspirated may not be noticeable by the operator. For liquids with higher viscosity, such as glycerol, serum, plasma, or polyethylene glycol, the delay can be appreciable (many seconds). If the tip is withdrawn from the source vessel prematurely, such as at time tx, then the desired volume of liquid will not be aspirated, which means that an error in the amount of liquid subsequently delivered will occur. (Moreover, if the tip is left immersed in the source vessel longer than is necessary, an error in the amount of liquid aspirated will occur due to excessive evaporation and heat transfer.)
As a more specific illustration of this problem, a typical manual 100 μL air displacement delivery device (e.g., an Eppendorf 100 μL Research Pipette, which is commercially available from Eppendorf AG of Hamburg, Germany) requires 33 seconds to fully aspirate a 100 μL sample of anhydrous glycerol at room temperature. By the time 20 seconds have elapsed during this period, liquid flows so slowly into the tip, as judged by the location of the interface in the tip, that it is difficult to know when the aspiration is complete. Thirty-three seconds is a long time to wait, especially when the device is to be carefully held poised over the source vessel and the tip immersed to the correct depth (2-3 mm) for that entire time. Therefore, a busy operator often will cut that wait short by withdrawing the tip prematurely, which in turn will cause errors in liquid dispensation. Further exacerbating this problem, it often is very difficult for an operator to know how long to hold the tip within the source vessel, since this will depend on the viscosity of the particular liquid being aspirated.
It is often considered good practice for the operator to adjust the aspiration speed of a device based upon the viscosity of the liquid being aspirated. Most electronic devices are provided with a control capability for the operator to set the desired aspiration speed. It is, however, difficult for an operator to know the best speed for a given sample of liquid. For instance, there is wide variation in the viscosity of serum samples; an operator can hardly be expected to determine the best aspiration speed for each sample. Therefore, an operator who is about to aspirate a particular serum is more likely to set a value that he/she thinks may be adequate for that serum type and hope for the best. In doing so, he/she may be aspirate different volumes among samples due to the sources of error already described.
Further, due to surface properties of the aspirated liquid, movement of the piston of these devices will not cause the interface meniscus to move immediately. If no liquid is in the tip, a certain negative pressure of air 5 at the interface 5′ is required to overcome the surface tension of the liquid and draw it into the tip. Thus, in this case, no liquid is drawn into the tip while the surface tension is being overcome. This failure to initially aspirate any liquid causes a small error in the amount of liquid aspirated by the device. For an existing device, this error is accounted for whenever the device is sent for calibration. For these devices, it turns out that so long as the type of liquid aspirated during use is the same as the type of liquid aspirated during this calibration process, which is rarely the case, the error is adequately compensated. If, however, the device is used to deliver liquid having a different surface tension than the liquid aspirated in the calibration process, which is often the case, then the correction introduced during calibration will not be correct, and therefore an error in delivery volume will result.
Likewise, when dispensing liquid from the tip, the first motion of the piston creates a slight overpressure of air 5 at the interface 5′, yet results in no motion of the interface meniscus. This is true because the contact angle between the liquid and an inner wall of the tip changes as pressure is applied, and this in turn causes a change in the shape of the interface meniscus without causing the point of contact between liquid and inner wall of the tip to move. This will be true for any liquid-solid interface for which the leading contact angle is greater than the trailing contact angle. It is a stick-slip phenomenon. When the overpressure becomes great enough, the interface meniscus will start to move along the inner wall of the tip. Additionally, if the tip is held in ambient air as liquid is dispensed, then surface tension of the liquid will tend to cause liquid to be held in the tip until enough overpressure is created to overcome the forces of surface tension and stick-slip at open end 7′ of FIGS. 1 and 2 of the tip, thereby allowing the liquid to exit the tip. In the instance that all of the liquid in the tip is being dispensed at once, these effects are irrelevant. It is only necessary to produce enough piston motion to dispense all of the liquid, including providing a “blowout” through the tip opening 7′ to expel the final droplet of liquid. In the instance that multiple dispense operations are to be performed following a single aspiration, it is desirable that the volume of each dispense be the same. Very commonly, in practice, the first such dispense will be a different volume than the others, and, for that reason, should be discarded.
As liquid is aspirated into the tip, a negative pressure is required within the tip to hold the interface meniscus above the level of the liquid in the source vessel because of the force of gravity on the liquid in the tip. This pressure is usually called the “head pressure”, and it is dependent on the density of the liquid aspirated. Usually, when the device is sent for calibration, the effect of this head pressure is corrected. If, however, the density of the liquid used to calibrate the device is different than the density of the liquid being delivered in another operation of the device, an error in delivered volume due to liquid density will be seen. According to ISO 8655 part 2, Appendix A, the magnitude of such error can be up to 1%.
Another application of the liquid delivery device is to use it in a titration procedure, in which the operator adds one liquid to another in small increments, while observing by eye or instrument the process of a reaction. For instance, the acidity of a sample of liquid can be determined by adding a base of known concentration and an indicator dye. In these applications, the base is added in small aliquots while stirring the liquids together and observing the color of the solution. When pH reaches a particular value, the dye changes color, signaling the operator to stop adding basic solution. It often is desirable to know the volume of base that was added to change the pH to that point. Using a device such as shown in FIGS. 1 and 2, the volume of liquid added is dependent on the density and surface properties of the liquid, as explained above. Calibration of the device with water does not provide the proper correction when the device is actually used to dispense another liquid type.
As might be expected, the best reproducibility of results from using a liquid delivery device is obtained when the pause between the end of aspiration and the removal of the tip from the source vessel is not too short or not too long. This can be determined by experimentation and experience for a device of a particular size or design, as well as based upon the type of liquid to be aspirated. A several second pause is generally recommended for aqueous solutions; however this is a generalization that may not be optimal for all situations.
When aspirating liquids with high vapor pressure, the error created due to heat transfer between the liquid and the interface is likely to be much less than the error due to evaporation of the liquid. For this reason, it is generally best to precondition the tip and rest of the device very thoroughly, by saturating the air interface with vapor from the liquid, before quantitatively aspirating the liquid. It is nonetheless difficult for a user to know to what extent the tip and rest of the device must be preconditioned to avoid error. In these instances, the operator must balance the need to precondition the device versus time. Since operators often will err on the side of over preconditioning the device, much time can be needlessly wasted.
In the instance of existing automated liquid delivery devices 10 that are pre-programmed to follow a certain sequence, a given aspiration protocol may be optimal for one volume or liquid sample, but not for another. The person creating the protocol must experiment to determine the correct aspiration speed and pause interval, as well as the correct dispense speed and pause interval. Considerable time and expertise are required to find the best parameters to optimize the accuracy and precision of results.
In light of the above mentioned limitations of the existing air displacement delivery devices, what is needed, therefore, is an air displacement delivery system that is capable of optimizing its own performance based on one or more properties that it determines regarding the air contained within the apparatus, the ambient air surrounding the apparatus, and/or the type of liquid to be aspirated and/or dispensed. The needed system should minimize the length of time that its tip is immersed in a source vessel, and which minimizes the need to precondition its tip to attain accurate results. The needed system also should minimize oscillation by avoiding abrupt flow transitions by tapering off the flow of liquid at the end of aspiration, and should be able to detect the end of oscillation and determine when it is the best time to withdraw the tip from a source vessel. The needed system further should shorten the amount of time a tip must be held within a liquid sample after aspiration. Even further, the needed system should be able to determine viscosity of an aspirated liquid sample, and based on this viscosity value, dispense the most accurate and reproducible delivery volume in the least amount of time. Still further, the needed system should be able to determine the actual volume of an aspirated liquid aliquot, regardless of any surface tension between the system and the liquid sample from which the liquid aliquot is being aspirated. Still further, the needed system should be able to determine the actual volume of a liquid that is dispensed from the system during any given dispensation, regardless of any surface tension between the system and the surface of the liquid into which the system is to dispense or contact angles of that liquid. Still further, the needed system should be able to deliver a precise volume of a liquid independent of the actual density of that liquid. Still further, the needed system should be able to determine optimum pause time, i.e., the time between the end of aspiration and the removal of the tip from a liquid sample after aspiration. Still further, the needed system should be able to use information specific to the nature of the liquid to be dispensed for the purpose of minimizing dispensation error and dispense time.